Skp1, an essential subunit of the major SCF-class of ubiquitin (Ub)-ligases important for the turnover of major regulatory proteins, is subject to complex glycosylation in the cytoplasm of the social amoeba Dictyostelium. A project to define the pathway responsible for this unusual modification showed that it depends on an O2-sensitive cytoplasmic prolyl 4-hydroxylase (P4H1). P4H1 is an ortholog of mammalian enzymes proposed to serve as O2-sensors that regulate hypoxic responses of cells via the transcriptional factor subunit hypoxia inducible factor-alpha. Disruption of Dictyostelium P4H1 inhibits an O2-dependent step in terminal differentiation (culmination) suggesting that P4H1 is a critical oxygen-sensor in Dictyostelium that influences the activity of E3(SCF)Ub-ligases that control culmination. P4H1-null cells also apparently fail to induce prestalk cell tip organizer cells that regulate culmination. Using Dictyostelium as a model system for animals and pathogenic protists including Toxoplasma gondii, which informatics and biochemical studies suggest share this regulatory pathway, we will explore the following key predictions of the hypoxic regulation model: 1) P4H1 is expressed in and active in prestalk cell tip organizer cells, where it mediates O2- dependent modification of Skp1 and nuclear accumulation of Skp1. 2) Hyperactive P4H1 mutants isolated by directed evolution in a suppressor screen for hypoxic spore-formers will reveal a direct role of O2-binding in vivo. 3A) Mutation of the Skp1 hydroxylation site will recapitulate the phenotype of P4H1-disruption. 3B) SCF complex subunits will influence Skp1 hydroxylation/glycosylation in vitro. 3C) Skp1 hydroxylation/glycosylation will influence SCF complex formation in vivo. 4) GlcNAc-capping of hydroxylated Skp1 is essential for P4H1 function. The results will provide new infomration about the mechanism of O2- stimulation of P4H1 that is expected to be applicable to understanding of mammalian hypoxic regulation which is important for cancer, inflammation, and wound repair. The findings are expected to define key new steps of a novel mechanism for hypoxic regulation that will be applicable to protozoan parasites and possibly also invertebrate and/or mammalian animals. Based on the new research, drug discovery initiatives for animal P4H enzymes might find unanticipated applications in control of protozoan infectious disease, as inhibitors might compromise pathogen adaptation to hypoxic conditions or stress!